Interferometric Biosensor for High Sensitive Label-Free Recording of HiPS Cardiomyocytes Contraction in Vitro.

Heart disease remains a leading cause of global mortality, underscoring the need for advanced technologies to study cardiovascular diseases and develop effective treatments. We introduce an innovative interferometric biosensor for high-sensitivity and label-free recording of human induced pluripotent stem cell (hiPSC) cardiomyocyte contraction in vitro. Using an optical cavity, our device captures interference patterns caused by the contraction-induced displacement of a thin flexible membrane. First, we demonstrate the capability to quantify spontaneous contractions and discriminate between contraction and relaxation phases. We calculate a contraction-induced vertical membrane displacement close to 40 nm, which implies a traction stress of 34 ± 4 mN/mm2. Finally, we investigate the effects of a drug compound on contractility amplitude, revealing a significant reduction in contractile forces. The label-free and high-throughput nature of our biosensor may enhance drug screening processes and drug development for cardiac treatments. Our interferometric biosensor offers a novel approach for noninvasive and real-time assessment of cardiomyocyte contraction.

Long-term in vitro recording of cardiac action potentials on microelectrode arrays for chronic cardiotoxicity assessment.

The reliable identification of chronic cardiotoxic effects in in vitro screenings is fundamental for filtering out toxic molecular entities before in vivo animal experimentation and clinical trials. Present techniques such as patch-clamp, voltage indicators, and standard microelectrode arrays do not offer at the same time high sensitivity for measuring transmembrane ion currents and low-invasiveness for monitoring cells over long time. Here, we show that optoporation applied to microelectrode arrays enables measuring action potentials from human-derived cardiac syncytia for more than 1 continuous month and provides reliable data on chronic cardiotoxic effects caused by known compounds such as pentamidine. The technique has high potential for detecting chronic cardiotoxicity in the early phases of drug development.

Improving reliability and reducing costs of cardiotoxicity assessments using laser-induced cell poration on microelectrode arrays.

Drug-induced cardiotoxicity is a major barrier to drug development and a main cause of withdrawal of marketed drugs. Drugs can strongly alter the spontaneous functioning of the heart by interacting with the cardiac membrane ion channels. If these effects only surface during in vivo preclinical tests, clinical trials or worse after commercialization, the societal and economic burden will be significant and seriously hinder the efficient drug development process. Hence, cardiac safety pharmacology requires in vitro electrophysiological screening assays of all drug candidates to predict cardiotoxic effects before clinical trials. In the past 10 years, microelectrode array (MEA) technology began to be considered a valuable approach in pharmaceutical applications. However, an effective tool for high-throughput intracellular measurements, compatible with pharmaceutical standards, is not yet available. Here, we propose laser-induced optoacoustic poration combined with CMOS-MEA technology as a reliable and effective platform to detect cardiotoxicity. This approach enables the acquisition of high-quality action potential recordings from large numbers of cardiomyocytes within the same culture well, providing reliable data using single-well MEA devices and single cardiac syncytia per each drug. Thus, this technology could be applied in drug safety screening platforms reducing times and costs of cardiotoxicity assessments, while simultaneously improving the data reliability.

Nonylphenol acts on prostate adenocarcinoma cells via estrogen molecular pathways.

Estrogens play a role in the patho-physiology of the prostate. In the present work we studied the effects of nonylphenol (NP), a xenoestrogen, on human adenocarcinoma prostate cells (LNCaP). In order to understand molecular and cellular involvement, we observed the effects on cell cycle and we investigated the expression and the cellular localization of estrogen receptors and gene expression of cyclin D1, ki-67, c-myc, IL-8, IL-1ß. We performed the same experiments with 17ß-estradiol (E2), the most abundant estrogen circulating in nonpregnant humans in order to compare these two different substances. We demonstrated the ability of 1 × 10-10 M NP to induce proliferation of LNCaP, S-phase progression, increase of ERα expression and its translocation from the cytoplasm to the nucleus. Moreover, we observed an up-regulation of key target genes involved in cell cycle and inflammation process. Particularly, after NP treatment, IL-8 and IL-1ß mRNA levels are increased more than 50% indicating a major NP involvement in inflammation processes than E2. These data suggest the proliferative effects of NP on prostate adenocarcinoma cells and highlight some aspects of molecular pathways involved in prostate responses to NP.

Expression and potential role of the peptide orexin-A in prostate cancer.

The peptides orexin-A and orexin-B and their G protein-coupled OX1 and OX2 receptors are involved in multiple physiological processes in the central nervous system and peripheral organs. Altered expression or signaling dysregulation of orexins and their receptors have been associated with a wide range of human diseases including narcolepsy, obesity, drug addiction, and cancer. Although orexin-A, its precursor molecule prepro-orexin and OX1 receptor have been detected in the human normal and hyperplastic prostate tissues, their expression and function in the prostate cancer (PCa) remains to be addressed. Here, we demonstrate for the first time the immunohistochemical localization of orexin-A in human PCa specimens, and the expression of prepro-orexin and OX1 receptor at both protein and mRNA levels in these tissues. Orexin-A administration to the human androgen-dependent prostate carcinoma cells LNCaP up-regulates OX1 receptor expression resulting in a decrease of cell survival. Noteworthy, nanomolar concentrations of the peptide counteract the testosterone-induced nuclear translocation of the androgen receptor in the cells: the orexin-A action is prevented by the addition of the OX1 receptor antagonist SB-408124 to the test system. These findings indicate that orexin-A/OX1 receptor interaction interferes with the activity of the androgen receptor which regulates PCa onset and progression, thus suggesting that orexin-A and its receptor might represent novel therapeutic targets to challenge this aggressive cancer.

Mirroring Action Potentials: Label-Free, Accurate, and Noninvasive Electrophysiological Recordings of Human-Derived Cardiomyocytes.

The electrophysiological recording of action potentials in human cells is a long-sought objective due to its pivotal importance in many disciplines. Among the developed techniques, invasiveness remains a common issue, causing cytotoxicity or altering unpredictably cell physiological response. In this work, a new approach for recording intracellular signals of outstanding quality and with noninvasiveness is introduced. By taking profit of the concept of mirror charge in classical electrodynamics, the new proposed device transduces cell ionic currents into mirror charges in a microfluidic chamber, thus realizing a virtual mirror cell. By monitoring mirror charge dynamics, it is possible to effectively record the action potentials fired by the cells. Since there is no need for accessing or interacting with the cells, the method is intrinsically noninvasive. In addition, being based on optical recording, it shows high spatial resolution and high parallelization. As shown through a set of experiments, the presented methodology is an ideal candidate for the next generation devices for the reliable assessment of cardiotoxicity on human-derived cardiomyocytes. More generally, it paves the way toward a new family of in vitro biodevices that will lay a new milestone in the field of electrophysiology.

All-Optical and Label-Free Stimulation of Action Potentials in Neurons and Cardiomyocytes by Plasmonic Porous Metamaterials.

Optical stimulation technologies are gaining great consideration in cardiology, neuroscience studies, and drug discovery pathways by providing control over cell activity with high spatio-temporal resolution. However, this high precision requires manipulation of biological processes at genetic level concealing its development from broad scale application. Therefore, translating these technologies into tools for medical or pharmacological applications remains a challenge. Here, an all-optical nongenetic method for the modulation of electrogenic cells is introduced. It is demonstrated that plasmonic metamaterials can be used to elicit action potentials by converting near infrared laser pulses into stimulatory currents. The suggested approach allows for the stimulation of cardiomyocytes and neurons directly on commercial complementary metal-oxide semiconductor microelectrode arrays coupled with ultrafast pulsed laser, providing both stimulation and network-level recordings on the same device.

Intracellular action potential recordings from cardiomyocytes by ultrafast pulsed laser irradiation of fuzzy graphene microelectrodes.

Graphene with its unique electrical properties is a promising candidate for carbon-based biosensors such as microelectrodes and field effect transistors. Recently, graphene biosensors were successfully used for extracellular recording of action potentials in electrogenic cells; however, intracellular recordings remain beyond their current capabilities because of the lack of an efficient cell poration method. Here, we present a microelectrode platform consisting of out-of-plane grown three-dimensional fuzzy graphene (3DFG) that enables recording of intracellular cardiac action potentials with high signal-to-noise ratio. We exploit the generation of hot carriers by ultrafast pulsed laser for porating the cell membrane and creating an intimate contact between the 3DFG electrodes and the intracellular domain. This approach enables us to detect the effects of drugs on the action potential shape of human-derived cardiomyocytes. The 3DFG electrodes combined with laser poration may be used for all-carbon intracellular microelectrode arrays to allow monitoring of the cellular electrophysiological state.

Intracellular Recording of Human Cardiac Action Potentials on Market-Available Multielectrode Array Platforms.

High quality attenuated intracellular action potentials from large cell networks can be recorded on multi-electrode arrays by means of 3D vertical nanopillars using electrical pulses. However, most of the techniques require complex 3D nanostructures that prevent the straightforward translation into marketable products and the wide adoption in the scientific community. Moreover, 3D nanostructures are often delicate objects that cannot sustain several harsh use/cleaning cycles. On the contrary, laser optoacoustic poration allows the recording of action potentials on planar nanoporous electrodes made of noble metals. However, these constraints of the electrode material and morphology may also hinder the full exploitation of this methodology. Here, we show that optoacoustic poration is also very effective for porating cells on a large family of MEA electrode configurations, including robust electrodes made of nanoporous titanium nitride or disordered fractal-like gold nanostructures. This enables the recording of high quality cardiac action potentials in combination with optoacoustic poration, providing thus attenuated intracellular recordings on various already commercial devices used by a significant part of the research and industrial communities.

Valproic Acid Synergizes With Cisplatin and Cetuximab in vitro and in vivo in Head and Neck Cancer by Targeting the Mechanisms of Resistance.

Recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC) is a devastating malignancy with a poor prognosis. The combination of cisplatin (CDDP) plus cetuximab (CX) is one of the standard first-line treatments in this disease. However, this therapeutic regimen is often associated with high toxicity and resistance, suggesting that new combinatorial strategies are needed to improve its therapeutic index. In our study, we evaluated the antitumor effects of valproic acid (VPA), a well-known antiepileptic agent with histone deacetylase inhibitory activity, in combination with CDDP/CX doublet in head and neck squamous cell carcinoma (HNSCC) models. We demonstrated, in HNSCC cell lines, but not in normal human fibroblasts, that simultaneous exposure to equitoxic doses of VPA plus CDDP/CX resulted in a clear synergistic antiproliferative and pro-apoptotic effects. The synergistic antitumor effect was confirmed in four different 3D-self-assembled spheroid models, suggesting the ability of the combined approach to affect also the cancer stem cells compartment. Mechanistically, VPA enhanced DNA damage in combination treatment by reducing the mRNA expression of ERCC Excision Repair 1, a critical player in DNA repair, and by increasing CDDP intracellular concentration via upregulation at transcriptional level of CDDP influx channel copper transporter 1 and downregulation of the ATPAse ATP7B involved in CDDP-export. Valproic acid also induced a dose-dependent downregulation of epidermal growth factor receptor (EGFR) expression and of MAPK and AKT downstream signaling pathways and prevent CDDP- and/or CX-induced EGFR nuclear translocation, a well-known mechanism of resistance to chemotherapy. Indeed, VPA impaired the transcription of genes induced by non-canonical activity of nuclear EGFR, such as cyclin D1 and thymidylate synthase. Finally, we confirmed the synergistic antitumor effect also in vivo in both heterotopic and orthotopic models, demonstrating that the combined treatment completely blocked HNSCC xenograft tumors growth in nude mice. Overall, the introduction of a safe and generic drug such as VPA into the conventional treatment for R/M HNSCC represents an innovative and feasible antitumor strategy that warrants further clinical evaluation. A phase II clinical trial exploring the combination of VPA and CDDP/CX in R/M HNSCC patients is currently ongoing in our institute.

gH625-liposomes as tool for pituitary adenylate cyclase-activating polypeptide brain delivery.

The blood-brain barrier (BBB) regulates the traffic of molecules into the central nervous system (CNS) and also limits the drug delivery. Due to their flexible properties, liposomes are an attractive tool to deliver drugs across the BBB. We previously characterized gH625, a peptide derived from Herpes simplex virus 1. The present study investigates the efficiency of liposomes functionalized on their surface with gH625 to promote the brain uptake of neuroprotective peptide PACAP (pituitary adenylate cyclase-activating polypeptide). Using a rat in vitro BBB model, we showed that the liposomes preparations were non-toxic for the endothelial cells, as assessed by analysis of tight junction protein ZO1 organization and barrier integrity. Next, we found that gH625 improves the transfer of liposomes across endothelial cell monolayers, resulting in both low cellular uptake and increased transport of PACAP. Finally, in vivo results demonstrated that gH625 ameliorates the efficiency of liposomes to deliver PACAP to the mouse brain after intravenous administration. gH625-liposomes improve both PACAP reaching and crossing the BBB, as showed by the higher number of brain cells labelled with PACAP. gH625-liposomes represent a promising strategy to deliver therapeutic agents to CNS and to provide an effective imaging and diagnostic tool for the brain.

Enhanced uptake of gH625 by blood brain barrier compared to liver in vivo: characterization of the mechanism by an in vitro model and implications for delivery.

We have investigated the crossing of the blood brain barrier (BBB) by the peptide gH625 and compared to the uptake by liver in vivo. We clearly observed that in vivo administration of gH625 allows the crossing of the BBB, although part of the peptide is sequestered by the liver. Furthermore, we used a combination of biophysical techniques to gain insight into the mechanism of interaction with model membranes mimicking the BBB and the liver. We observed a stronger interaction for membranes mimicking the BBB where gH625 clearly undergoes a change in secondary structure, indicating the key role of the structural change in the uptake mechanism. We report model studies on liposomes which can be exploited for the optimization of delivery tools.

Polystyrene nanoparticles internalization in human gastric adenocarcinoma cells.

The increase in the use of nanoparticles, as a promising tool for drug delivery or as a food additive, raises questions about their interaction with biological systems, especially in terms of evoked responses. In this work, we evaluated the kinetics of uptake of 44 nm (NP44) and 100 nm (NP100) unmodified polystyrene nanoparticles (PS-NPs) in gastric adenocarcinoma (AGS) cells, as well as the endocytic mechanism involved, and the effect on cell viability and gene expression of genes involved in cell cycle regulation and inflammation processes. We showed that NP44 accumulate rapidly and more efficiently in the cytoplasm of AGS compared to NP100; both PS-NPs showed an energy dependent mechanism of internalization and a clathrin-mediated endocytosis pathway. Dose response treatments revealed a non-linear curve. PS-NPs also affected cell viability, inflammatory gene expression and cell morphology. NP44 strongly induced an up-regulation of IL-6 and IL-8 genes, two of the most important cytokines involved in gastric pathologies. Our study suggests that parameters such as time, size and concentration of NPs must be taken carefully into consideration during the development of drug delivery systems based on NPs and for the management of nanoparticles associated risk factors.

Catestatin and GABA(A)R related feeding habits rely on dopamine, ghrelin plus leptin neuroreceptor expression variations.

Catestatin (CST), an endogenously small sympathoinhibitory peptide is capable of interfering with the major cerebral neuroreceptor-blocking site, i.e. γ-aminobutyric acidA receptor (GABAAR) system especially in limbic brain areas that are involved with feeding behaviors. The GABAARergic-related effects seem to derive from its interaction with other molecular neuroreceptors such as dopaminergic, ghrelin and leptinergic. In this context, the present study aimed to investigate probable feeding responses (eating and drinking) induced by treatment with CST and the GABAAR antagonist bicucullin (BIC) alone or simultaneously (CST+BIC) in the Syrian hibernating hamster (Mesocricetus auratus) model. Hamsters that received these compounds via intracerebroventricular infusions displayed notable variations of feeding and drinking bouts. In particular, an anorexigenic response was evident following treatment with CST while BIC evoked a significant increase of eating and drinking behaviors. Surprisingly when both agents were given simultaneously, a predominating anorexigenic response was detected as shown by evident CST-dependent reduction of feeding bouts. Contextually such behaviors, especially those following the combined treatment were tightly correlated with the significantly increased cerebral dopamine receptor 1 (D1) plus reduced ghrelin receptor (GhsR) and leptin receptor (LepR) transcript levels. Overall, the anorexigenic effect of CST deriving from its tight interaction with GABAARs activity plus D1 and GhsR transcripts tends to propose these neuronal elements as pivotal factors responsible for feeding disorders.

Peptide gH625 enters into neuron and astrocyte cell lines and crosses the blood-brain barrier in rats.

Peptide gH625, derived from glycoprotein H of herpes simplex virus type 1, can enter cells efficiently and deliver a cargo. Nanoparticles armed with gH625 are able to cross an in vitro model of the blood-brain barrier (BBB). In the present study, in vitro experiments were performed to investigate whether gH625 can enter and accumulate in neuron and astrocyte cell lines. The ability of gH625 to cross the BBB in vivo was also evaluated. gH625 was administered in vivo to rats and its presence in the liver and in the brain was detected. Within 3.5 hours of intravenous administration, gH625 can be found beyond the BBB in proximity to cell neurites. gH625 has no toxic effects in vivo, since it does not affect the maximal oxidative capacity of the brain or the mitochondrial respiration rate. Our data suggest that gH625, with its ability to cross the BBB, represents a novel nanocarrier system for drug delivery to the central nervous system. These results open up new possibilities for direct delivery of drugs into patients in the field of theranostics and might address the treatment of several human diseases.

Nonylphenol effects on the HPA axis of the bioindicator vertebrate, Podarcis sicula lizard.

Nonylphenol (NP) is an endocrine disruptor widely distributed in the environment. It accumulates in the lipids of living organisms and enters the human food chain. The main source of human exposure is expected to be food, drinking water and foodstuff contaminated through leaching from packaging or pesticide formulation applications. NP acts as an estrogenic compound and it is able to mimic the action of estradiol 17ß (E2) by binding to the estrogen receptor (ER). The aim of the present study was to investigate the NP effects on the hypothalamic-pituitary-adrenal gland (HPA) axis of the bioindicator Podarcis sicula lizard. A time-dependent stimulation of the HPA axis and variations of both catecholamine plasma levels were showed. Moreover, NP effects on adrenal gland morphology were evaluated by light and transmission electron microscopy. Clear morphological signs of adrenal gland stimulation such as an increase of steroidogenic cord diameter and vascularization, a strong escalation of adrenaline cell number and a decrease of noradrenaline cells were observed. The notably elevated levels of adrenal hormones suggested a permanent turning on of hypothalamic corticotropin releasing factor (CRF) secretion together with a lack of the negative feedback of HPA axis, perturbing systemic responses of the organism. Our data may help to predict the biological alterations induced by NP and to extend its impact upon adrenal function.